Method of Treating or Retarding the Development of Blindness

ABSTRACT

A method for treating an ocular disorder characterized by the defect or absence of a normal gene in the ocular cells of a human or animal subject involves administering to the subject by subretinal injection an effective amount of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the normal gene under the control of a promoter sequence which expresses the product of the gene in the ocular cells. The ocular cells are preferably retinal pigment epithelial (RPE) cells, and the gene is preferably an RPE-specific gene, e.g., RPE65. The promoter is one that can express the gene product in the RPE cells. Compositions for subretinal administration are useful in this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/310,015 filed Jun. 20, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/766,267, filed Feb. 13, 2013 (now abandoned),which is a continuation of U.S. patent application Ser. No. 13/406,666,filed Feb. 28, 2012, now abandoned, which is a continuation of U.S.patent application Ser. No. 12/832,282, filed Jul. 8, 2010, now U.S.Pat. No. 8,147,823, issued Apr. 3, 2012, which is a continuation of U.S.patent application Ser. No. 12/253,955, filed Oct. 18, 2008, nowabandoned, which is a continuation of U.S. patent application Ser. No.11/511,201, filed Aug. 28, 2006, now abandoned, which is a continuationof U.S. patent application Ser. No. 10/300,720, filed Nov. 20, 2002, nowabandoned, which is a continuation of International Patent ApplicationNo. PCT/US02/11314, filed Apr. 11, 2002, now expired, which claims thebenefit of the priority of U.S. Provisional Patent Application No.60/283,766, filed Apr. 13, 2001, now expired, which applications areincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.EY010820, EY011123, EY006855, NS036202, EY011142, and EY013132 awardedby the National Institutes of Health. The government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

The invention relates generally to the use of recombinant viruses todeliver a desired transgene to retinal pigment epithelial cells ofpatients suffering from retinal degenerative diseases.

The relationship between the neurosensory photoreceptors and theadjacent retinal pigment epithelium (RPE) controls not only normalretinal function, but also the pathogenesis of hereditary retinaldegenerations. Recent progress has identified the molecular bases forprimary photoreceptor diseases, such as retinitis pigmentosa (Dryja, T.P., et al. 1990 Nature 343, 364-366; Farrar, G. J., et al. 1991 Nature354, 478-480; and McLaughlin, M. E. et al, 1993 Nature Genetics 4,130-134). Similarly the molecular bases for RPE diseases that causephotoreceptor blindness, such as child-onset severe retinal dystrophy,Leber's congenital amaurosis, and Best macular dystrophy, have beenidentified (Gu, S. -M., et al. 1997 Nature Genetics 17, 194-197;Marlhens, F., et al. 1997 Nature Genetics 17, 139-141; Petrukin, K., etal. 1998 Nature Genet 19, 241-247; and D'Cruz, P., et al. 2000 Hum. Mol.Genet. 9, 645-651). Despite these reported scientific advances,effective therapy for human retinal degenerations is still lacking.

Retinal gene therapy has been considered a possible therapeutic optionfor man. For example, U.S. Pat. No. 5,827,702 refers to methods forgenerating a genetically engineered ocular cell by contacting the cellwith an exogenous nucleic acid under conditions in which the exogenousnucleic acid is taken up by the cell for expression. The exogenousnucleic acid is described as a retrovirus, an adenovirus, anadeno-associated virus or a plasmid. See, also, International PatentPublication Nos. WO 00/15822, published Mar. 23, 2000 and WO 98/48097,published Oct. 29, 1998.

A review of gene therapy efforts to date indicates that such effortshave focused mainly on slowing down retinal degeneration in rodentmodels of primary photoreceptor diseases. Normal genes andmutation-specific ribozymes delivered to photoreceptors have prolongedthe lifetime of these cells otherwise doomed for apoptotic cell death(Bennett, J., et al. 1996 Nat. Med. 2, 649-654; Bennett, J., et al. 1998Gene Therapy 5, 1156-1164; Kumar-Singh, R. & Farber, D., 1998 Hum. Mol.Genet. 7, 1893-900; Lewin, A. S., et al. 1998 Nat. Med. 4, 967-971; Ali,R., et al. 2000 Nat. Genet. 25, 306-310; Takahashi, M. et al, 1999 J.Virol. 73, 7812-6; Lau, D., et al. 2000 Invest. Ophthalmol. Vis. Sci.41, 3622-3633; and LaVail, M. M., et al. 2000 Proc Natl Acad Sci USA 97,11488-11493).

Retinal gene transfer of a reporter gene, green fluorescent protein,using a recombinant adeno-associated virus was demonstrated in normalprimates (Bennett, J., et al. 1999 Proc. Natl. Acad. Sci. USA 96,9920-9925). However, an as-yet unmet goal of research is the restorationof vision in a blinding disease of animals, particularly humans andother mammals, caused by genetic defects in RPE and/or photoreceptorcells.

There remains a need in the art for methods for effectively treatinghumans and other mammals or other animals suffering from blindness dueto genetic defects or deficiencies, so as to restore sufficient visionto enable the subject to function in response to visual cues.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for treating an oculardisorder in a human or animal subject characterized by the defect orabsence of a normal gene in the ocular cells. The method includesadministering to the subject by subretinal injection an effective amountof a recombinant adeno-associated virus carrying a nucleic acid sequenceencoding the normal gene under the control of a promoter sequence whichexpresses the product of the gene in the ocular cells.

In another aspect, the invention provides a method for treating anocular disorder in a human or animal subject characterized by the defector absence of a normal gene in the retinal pigment epithelial (RPE)cells of the subject. The method involves administering to the subjectby subretinal injection an effective amount of a recombinant viruscarrying a nucleic acid sequence encoding a normal retinal pigmentepithelial (RPE) cell-specific gene under the control of a promotersequence which expresses the product of the gene in RPE cells. In oneembodiment, the gene is the RPE65 gene.

In another aspect, the invention provides a method for treating Lebercongenital amaurosis in a subject by administering to the subject bysubretinal injection an effective amount of a recombinant virus carryinga nucleic acid sequence encoding a normal gene under the control of apromoter sequence which expresses the product of the gene in ocularcells, wherein the cells contain a mutated version of the gene.Expression of the normal gene provides to the cells the productnecessary to restore or maintain vision in the subject. In oneembodiment, the cells are RPE or photoreceptor cells, and the promotersare cell-specific promoters.

In still another embodiment, the invention provides a composition fortreatment of an ocular disorder characterized by the defect or absenceof a normal gene in the ocular cells of the subject. Such compositionscomprise effective amounts of a recombinant adeno-associated viruscarrying a nucleic acid sequence encoding the normal gene under thecontrol of a promoter sequence which expresses the product of the genein the ocular cells, formulated with a carrier and additional componentssuitable for subretinal injection. In one embodiment, the normal gene isRPE65.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for treating an ocular disorder in ahuman, other mammalian or other animal subject. In particular, theocular disorder is one which involves a mutated or absent gene in aretinal pigment epithelial cell or a photoreceptor cell. The method ofthis invention comprises the step of administering to the subject bysubretinal injection an effective amount of a recombinant virus carryinga nucleic acid sequence encoding an ocular cell-specific normal geneoperably linked to, or under the control of, a promoter sequence whichdirects the expression of the product of the gene in the ocular cellsand replaces the lack of expression or incorrect expression of themutated or absent gene.

A. The Ocular Disorder

In particular, this method is useful for the treatment and/orrestoration of at least partial vision to subjects that have lost visiondue to ocular disorders, such as RPE-associated retinopathies, which arecharacterized by a long-term preservation of ocular tissue structuredespite loss of function and by the association between function lossand the defect or absence of a normal gene in the ocular cells of thesubject. A variety of such ocular disorders are known, such as childhoodonset blinding diseases, retinitis pigmentosa, macular degeneration, anddiabetic retinopathy, as well as ocular blinding diseases known in theart. It is anticipated that these other disorders, as well as blindingdisorders of presently unknown causation which later are characterizedby the same description as above, may also be successfully treated bythis method. Thus, the particular ocular disorder treated by this methodmay include the above-mentioned disorders and a number of diseases whichhave yet to be so characterized. For purposes of illustration of thisinvention, the particular ocular disorder being treated in the examplesis Leber congenital amaurosis, which affects humans. However, thisinvention is not limited to the treatment of that disorder alone.

Leber congenital amaurosis (LCA) is a severe childhood-onset blindingdisease which can be caused by mutations in the retinal pigmentepithelium (RPE)-specific gene, RPE65. A naturally-occurring largeanimal model of an analogous severe disease of retinal degenerations isthe RPE65 mutant dog. LCA causes near total blindness from early inlife. Among the molecular causes of LCA are mutations in the geneencoding an RPE protein, RPE65. RPE65 is an evolutionarily-conserved65kDa membrane-associated protein (Redmond, T. & Hamel, C. 2000 Meth.Enzymol. 317, 705-724 and Bavik, C. et al, 1992 J. Biol. Chem. 267,23035-23042), which is important in retinoid metabolism (Saari, J. 2000Invest Ophthalmol Vis Sci 41, 337-348; Ma, J. -X. et al, 1998 J BiolChem 1443, 255-261; and Simon, A. et al, 1995 J Biol Chem 270,1107-1112). Currently there is no treatment for LCA and related earlyonset retinal degenerative diseases.

RPE65 deficiency in mice results in accumulation of all-trans-retinylesters, undetectable levels of rhodopsin, rod photoreceptor dysfunction,inclusions in the RPE, and slow retinal degeneration. The compound9-cis-retinal can restore visual pigment and function in RPE65-deficientmice (Redmond, T., et al. 1998 Nat. Genet 20, 344-351 and Van Hooser, J.P., et al. 2000 Proc. Natl Acad Sci USA 97, 8623-8628).

The RPE65 mutant dog shows early and severe visual impairment caused bya homozygous 4 bp-deletion in the RPE65 gene. The deletion results in aframe shift leading to a premature stop codon, eliminating more thantwo-thirds of the wildtype polypeptide. Histopathology in homozygotesshows prominent RPE inclusions and slightly abnormal rod photoreceptormorphology present within the first year of life, and slowly progressivephotoreceptor degeneration in older dogs. See, e.g., Wrigstad, A.Hereditary Dystrophy of the Retina and the Retinal Pigment Epithelium ina Strain of Briard Dogs: A Clinical, Morphological andElectrophysiological Study. Linkoping University Medical Dissertations(1994); Narfstrom, K. et al, 1989 Brit J Ophthalmol.

73, 750-756; and Aguirre, G., et al. 1998 Mol. Vis. 4, 23.

B. Vectors for Use in the Method

According to the various embodiments of the present invention, a varietyof known nucleic acid vectors may be used in these methods, e.g.,recombinant viruses, such as recombinant adeno-associated virus (AAV),recombinant adenoviruses, recombinant retroviruses, recombinantpoxviruses, and other known viruses in the art, as well as plasmids,cosmids and phages, etc. A wealth of publications known to those ofskill in the art discusses the use of a variety of such vectors fordelivery of genes (see, e.g., Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A. et al,2001 Nat. Medic., 7(1):33-40; and Walther W. and Stein U., 2000 Drugs,60(2):249-71). In one embodiment of this invention the vector is arecombinant AAV carrying a wildtype (i.e., normal) version of a selectedtransgene-encoding cDNA driven by a promoter that expresses the productof the wildtype cDNA in selected ocular cells of the affected subject.Methods for assembly of the recombinant vectors are well-known (see,e.g., International Patent Publication No. WO 00/15822, published Mar.23, 2000 and other references cited herein). To exemplify the methodsand compositions of this invention, the presently preferred vector, arecombinant AAV is described in detail.

1. AAV Vectors

Adeno-associated viruses are small, single-stranded DNA viruses whichrequire helper virus to facilitate efficient replication (K. I. Berns,Parvoviridae: the viruses and their replication, p. 1007-1041, in F. N.Fields et al., Fundamental Virology, 3rd ed., vol. 2, (Lippencott-RavenPublishers, Philadelphia, Pa.) (1995)). The 4.7 kb genome of AAV ischaracterized by two inverted terminal repeats (ITR) and two openreading frames which encode the Rep proteins and Cap proteins,respectively. The Rep reading frame encodes four proteins of molecularweight 78 kD, 68 kD, 52 kD and 40 kD. These proteins function mainly inregulating AAV replication and rescue and integration of the AAV into ahost cell's chromosomes. The Cap reading frame encodes three structuralproteins of molecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD (VP3)(Berns, cited above) which form the virion capsid. More than 80% oftotal proteins in AAV virion comprise VP3.

Flanking the rep and cap open reading frames at the 5′ and 3′ ends are145 by inverted terminal repeats (ITRs), the first 125 by of which arecapable of forming Y- or T-shaped duplex structures. The two ITRs arethe only cis elements essential for AAV replication, rescue, packagingand integration of the AAV genome. There are two conformations of AAVITRs called “flip” and “flop”. These differences in conformationoriginated from the replication model of adeno-associated virus whichuses the ITR to initiate and reinitiate the replication (R. O. Snyder etal, 1993, J. Virol., 67:6096-6104 (1993); K. I. Berns, 1990Microbiological Reviews, 54:316-329). The entire rep and cap domains canbe excised and replaced with a therapeutic or reporter transgene (B. J.Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press,pp.155-168 (1990)).

AAVs have been found in many animal species, including primates, canine,fowl and human (F. A. Murphy et al., “The Classification andNomenclature of Viruses: Sixth Report of the International Committee onTaxonomy of Viruses”, Archives of Virology, (Springer-Verlag, Vienna)(1995)). Six primate serotypes have been reported (AAV1, AAV2, AAV3,AAV4, AAV5 and AAV6). The AAV ITR sequences and other AAV sequencesemployed in generating the minigenes, vectors, and capsids, and otherconstructs used in the present invention may be obtained from a varietyof sources. For example, the sequences may be provided by AAV type 5,AAV type 2, AAV type 1, AAV type 3, AAV type 4, AAV type 6, or other AAVserotypes or other adenoviruses, including presently identified humanAAV types and AAV serotypes yet to be identified. Similarly, AAVs knownto infect other animals may also provide these ITRs employed in themolecules or constructs of this invention. Similarly, the capsids from avariety of serotypes of AAV may be “mixed and matched” with the othervector components. See, e.g., International Patent Publication No. WO01/83692, published Nov. 8, 2001, and incorporated herein by reference.A variety of these viral serotypes and strains are available from theAmerican Type Culture Collection, Manassas, Virginia, or are availablefrom a variety of academic or commercial sources. Alternatively, it maybe desirable to synthesize sequences used in preparing the vectors andviruses of the invention using known techniques, which may utilize AAVsequences which are published and/or available from a variety ofdatabases. The source of the sequences utilized in preparation of theconstructs of the invention, is not a limitation of the presentinvention. Similarly, the selection of the species and serotype of AAVthat provides these sequences is within the skill of the artisan anddoes not limit the following invention.

2. The Minigene

For use in the present invention, the AAV sequences are typically in theform of a rAAV construct (e.g., a minigene or cassette) which ispackaged into a rAAV virion. At a minimum, the rAAV minigene useful inthis invention is formed by AAV inverted terminal repeat sequences(ITRs) and a heterologous molecule for delivery to a host cell. Mostsuitably, the minigene contains AAV 5′ ITRs and 3′ ITRs located 5′ and3′ to the heterologous molecule, respectively. However, in certainembodiments, it may be desirable for the minigene to contain the 5′ ITRand 3′ ITR sequences arranged in tandem, e.g., 5′ to 3′ or ahead-to-tail, or in another alternative configuration. In still otherembodiments, it may be desirable for the minigene to contain multiplecopies of the ITRs or to have 5′ ITRs (or conversely, 3′ ITRs) locatedboth 5′ and 3′ to the heterologous molecule. The ITRs sequences may belocated immediately upstream and/or downstream of the heterologousmolecule, or there may be intervening sequences. The ITRs may beselected from AAV5, or from among the other AAV serotypes, as describedherein. Optionally, a minigene may contain 5′ ITRs from one serotype and3′ ITRs from a second serotype. The AAV sequences employed arepreferably the 145 by cis-acting 5′ and 3′ inverted terminal repeatsequences (See, e.g., B. J. Carter, cited above). Preferably, the entiresequences encoding the ITRs are used in the molecule, although somedegree of minor modification of these sequences is permissible. Theability to modify these ITR sequences is within the skill of the art.(See, e.g., texts such as Sambrook et al, “Molecular Cloning. ALaboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York(1989); Carter et al, cited above; and K. Fisher et al., 19961 Virol.,70:520-532). One of skill in the art can readily engineer the rAAV virusby methods known to the art (e.g., Bennett, J., et al. 1999 Proc. Natl.Acad. Sci. USA 96, 9920-9925). An example of such a molecule employed inthe present invention is a “cis-acting” plasmid containing theheterologous molecule flanked by the 5′ and 3′ AAV ITR sequences.

The heterologous molecule may be any substance which is desired to bedelivered to a cell, including, without limitation, a polypeptide,protein, enzyme, carbohydrate, chemical moiety, or nucleic acidsequences which may include oligonucleotides, RNA, and/or DNA.Preferably, for use in this invention, the heterologous molecule is aselected transgene under the control of a selected promoter and otherconventional vector regulatory components. See, e.g., U.S. Pat. Nos.5,856,152 and 5,871,982. In one embodiment, the heterologous moleculemay be a nucleic acid molecule which introduces specific geneticmodifications into human chromosomes, e.g., for correction of mutatedgenes. See, e.g., D. W. Russell & R. K. Hirata, 1998 Nat. Genet.,18:325-330.

a. The Transgene

In another desirable embodiment, the heterologous molecule is a nucleicacid molecule is a transgene. As used herein, “transgene” refers to anucleic acid sequence heterologous to the AAV sequence, encoding adesired product, e.g., a polypeptide or protein of interest, and theregulatory sequences which direct transcription and/or translationthereof in a host cell, and permit expression of the encoded product ina host cell. Suitable encoded products and regulatory sequences arediscussed in more detail below. However, the selection of theheterologous molecule delivered by the AAV minigene is not a limitationof the present invention.

In one embodiment of the method, where the ocular disorder is caused bya mutation in a normal retinal pigment epithelium (RPE)-specific gene,the ocular cells which are the target of the treatment method are theretinal pigment epithelial (RPE) cells. The specific gene which ismutated or absent in the disorder may be the RPE65 gene. Another genewhich is mutated or absent in the disorder in humans may be thearylhydrocarbon-interacting receptor protein like 1 (AIPL1). Thus, thenormal gene, i.e., the transgene, present in the recombinant virus isthe normal, species-matched version of the mutated gene, e.g., wildtypecanine RPE65 for the treatment of canine LCA or wildtype human RPE65 forthe treatment of human LCA, wildtype human AIPL1 for the treatment of acertain type of human blinding diseases, etc. In still anotherembodiment, the gene can be the CRB1 (RP12) gene. In another embodiment,the transgene can be the lecithin retinal acetyltransferase (LRAT) gene.These transgenes, as well as other transgenes useful for delivery to theeye may be obtained from conventional sources, e.g., from universitylaboratories or depositories, or synthesized from information obtainedfrom Genbank by known techniques.

In another embodiment of the method, where the ocular disorder is causedby a mutation in a normal photoreceptor-specific gene, the ocular cellswhich are the target of the treatment method are the photoreceptorcells. The specific gene which is mutated or absent in the disorder maybe the photoreceptor-specific homeo box gene (CRX). Alternatively, thespecific gene which is mutated or absent in the disorder may be theretinal guanylate cyclase gene (GUCY2D). In still another embodiment,the transgene is a nucleotide sequence encoding RPGR Interacting Protein1 (RPGRIP1). Thus, the normal gene, i.e., the transgene, present in therecombinant adeno-associated virus is the normal, species-matchedversion of the mutated gene, e.g., wildtype murine CRX for the treatmentof the correlative murine blinding disorder or wildtype human CRX forthe treatment of the correlative human blinding disorder, wildtypechicken GUCY2D for the treatment of the correlative chicken blindingdisorder or wildtype human GUCY2D for the treatment of the correlativehuman blinding disorder, etc. These transgenes may be obtained fromconventional sources, e.g., from university laboratories ordepositories, or synthesized from information obtained from Genbank byknown techniques.

As discussed above, still other genes may be added to this list,including the LCA genes referred to as LCA3, located at chromosome 14q24and LCA5, located at chromosome 6q11-q16, among others.

Genes responsible for disorders other than LCA may also be employed asthe transgene, as suitable ocular diseases are identified. Thus,different transgene may be used in the design of similar vectors of thisinvention for the treatment of disorders other than LCA. Among the knowngenes which may be absent or mutated in the blinding disordersidentified above include dystrophin, ABCR, EMP1, TIMP3, MERTCK andELOVL4. One or more of the wildtypes of these genes may be administeredto ocular cells, particularly the RPE, in the same manner as is theexemplified RPE65 for the treatment of LCA. One of skill in the art mayobtain the appropriate gene sequences and design the appropriate vectorsfor such use in view of this disclosure and in view of other informationknown to the art.

In certain situations, a different transgene may be used to encode eachsubunit of a protein, or to encode different peptides or proteins. Thisis desirable when the size of the DNA encoding the protein subunit islarge, e.g., for an immunoglobulin, the platelet-derived growth factor,or a dystrophin protein. In order for the cell to produce themulti-subunit protein, a cell is infected with the recombinant viruscontaining each of the different subunits. In another embodiment,different subunits of a protein may be encoded by the same transgene. Inthis case, a single transgene includes the DNA encoding each of thesubunits, with the DNA for each subunit separated by an internalribozyme entry site (IRES). This is desirable when the size of the DNAencoding each of the subunits is small, e.g., total of the DNA encodingthe subunits and the IRES is less than five kilobases. Alternatively,other methods which do not require the use of an IRES may be used forco-expression of proteins. Such other methods may involve the use of asecond internal promoter, an alternative splice signal, a co- orpost-translational proteolytic cleavage strategy, among others which areknown to those of skill in the art.

b. Regulatory Sequences

The minigene or transgene includes appropriate sequences that areoperably linked to the nucleic acid sequences encoding the product ofinterest to promote its expression in a host cell. “Operably linked”sequences present in the minigene include both expression controlsequences (e.g. promoters) that are contiguous with the coding sequencesfor the product of interest and expression control sequences that act intrans or at a distance to control the expression of the product ofinterest. In addition to being useful in the transgene, the regulatoryelements described herein may also be used in other heterologousmolecules and the other constructs described in this application.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein processing and/or secretion. A great number of expressioncontrol sequences, e.g., native, constitutive, inducible and/ortissue-specific, are known in the art and may be utilized to driveexpression of the gene, depending upon the type of expression desired.

For eukaryotic cells, expression control sequences typically include apromoter, an enhancer, such as one derived from an immunoglobulin gene,SV40, cytomegalovirus, etc., and a polyadenylation sequence which mayinclude splice donor and acceptor sites. The polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ ITR sequence. In one embodiment, the bovine growth hormone polyA isused.

The regulatory sequences useful in the constructs of the presentinvention may also contain an intron, desirably located between thepromoter/enhancer sequence and the gene. One possible intron sequence isalso derived from SV-40, and is referred to as the SV-40 T intronsequence. Another suitable sequence includes the woodchuck hepatitisvirus post-transcriptional element. (See, e.g., L. Wang and I. Verma,1999 Proc. Natl. Acad. Sci., USA, 96:3906-3910).

Another regulatory component of the rAAV useful in the method of theinvention is an internal ribosome entry site (IRES). An IRES sequence,or other suitable systems as are discussed above, may be used to producemore than one polypeptide from a single gene transcript. An IRES (orother suitable sequence) is used to produce a protein that contains morethan one polypeptide chain or to express two different proteins from orwithin the same cell. An exemplary IRES is the poliovirus internalribosome entry sequence, which supports transgene expression inphotoreceptors, RPE and ganglion cells. Preferably, the IRES is located3=to the transgene in the rAAV vector.

The selection of the promoter to be employed in the rAAV may be madefrom among a wide number of constitutive or inducible promoters that canexpress the selected transgene in an ocular. In a preferred embodiment,the promoter is cell-specific. The term “cell-specific” means that theparticular promoter selected for the recombinant vector can directexpression of the selected transgene is a particular ocular cell type.As one example, the promoter is specific for expression of the transgenein RPE cells. As another example, the promoter is specific forexpression of the transgene in photoreceptor cells.

Examples of constitutive promoters which may be included in the rAAV ofthis invention include, without limitation, the exemplified CMVimmediate early enhancer/chicken β-actin (CβA) promoter-exon 1-intron 1element of Example 1, the RSV LTR promoter/enhancer, the SV40 promoter,the CMV promoter, the dihydrofolate reductase promoter, and thephosphoglycerol kinase (PGK) promoter.

RPE-specific promoters include, for example, the RPE-65 promoter, thetissue inhibitor of metalloproteinase 3 (Timp3) promoter, and thetyrosinase promoter. Still other RPE-specific promoters are known tothose of skill in the art. See, e.g., the promoters described inInternational Patent Publication No. WO 00/15822.

Examples of photoreceptor specific promoters include, withoutlimitation, the rod opsin promoter, the red-green opsin promoter, theblue opsin promoter, the inter photoreceptor binding protein (IRBP)promoter and the cGMP-β-phosphodiesterase promoter. See, e.g., thepromoters described in International Patent Publication No. WO 98/48097.

Alternatively, an inducible promoter is employed to express thetransgene product, so as to control the amount and timing of the ocularcell=s production thereof. Such promoters can be useful if the geneproduct proves to be toxic to the cell upon excessive accumulation.Inducible promoters include those known in the art and those discussedabove including, without limitation, the zinc-inducible sheepmetallothionine (MT) promoter; the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter; the T7 promoter; the ecdysoneinsect promoter; the tetracycline-repressible system; thetetracycline-inducible system; the RU486-inducible system; and therapamycin-inducible system. Any type of inducible promoter which istightly regulated and is specific for the particular target ocular celltype may be used. Other types of inducible promoters which may be usefulin this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, a particularlydifferentiation state of the cell, or in replicating cells only.

Selection of these and other common vector and regulatory elements areconventional and many such sequences are available. See, e.g., Sambrooket al, and references cited therein at, for example, pages 3.18-3.26 and16.17-16.27 and Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1989). Of course, not all vectors andexpression control sequences will function equally well to express allof the transgenes of this invention. However, one of skill in the artmay make a selection among these expression control sequences withoutdeparting from the scope of this invention. Suitable promoter/enhancersequences may be selected by one of skill in the art using the guidanceprovided by this application. Such selection is a routine matter and isnot a limitation of the molecule or construct. For instance, one mayselect one or more expression control sequences, operably link thesequence to a transgene of interest, and insert the “minigene”comprising the expression control sequence and the transgene into an AAVvector. The vector may be packaged into an infectious particle or virionfollowing one of the methods for packaging the rAAV taught in the art.

C. Production of the rAAV

The rAAV virus of the invention may be constructed and produced usingthe materials and methods described herein, as well as those known tothose of skill in the art. Such engineering methods used to constructany embodiment of this invention are known to those with skill innucleic acid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques. See, e.g., Sambrook et al, andAusubel et al., cited above; and International Patent Publication No. WO95/13598. Further, methods suitable for producing a rAAV cassette in anadenoviral capsid have been described in U.S. Pat. Nos. 5,856,152 and5,871,982.

Briefly, in order to package the rAAV construct into a rAAV virion, ahost cell must contain sequences necessary to express AAV rep and AAVcap or functional fragments thereof as well as helper genes essentialfor AAV production. For example, the rep78/52 proteins may be sufficientto provide the necessary rep functions. The AAV rep and cap sequencesare obtained from an AAV source as identified above. The AAV rep and capsequences may be introduced into the host cell in any manner known toone in the art as described above, including, without limitation,transfection, electroporation, liposome delivery, membrane fusiontechniques, high velocity DNA-coated pellets, viral infection andprotoplast fusion. In one embodiment, the rep and cap sequences may betransfected into the host cell by one or more nucleic acid molecules andexist stably in the cell as an episome. In another embodiment, the repand cap sequences are stably integrated into the genome of the cell.Another embodiment has the rep and cap sequences transiently expressedin the host cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

The rep and cap sequences, along with their expression controlsequences, may be supplied on a single vector, or each sequence may besupplied on its own vector. Preferably, the rep and cap sequences aresupplied on the same vector. Alternatively, the rep and cap sequencesmay be supplied on a vector that contains other DNA sequences that areto be introduced into the host cells. Preferably, the promoter used inthis construct may be any suitable constitutive, inducible or nativepromoters known to one of skill in the art. The molecule providing therep and cap proteins may be in any form which transfers these componentsto the host cell. Desirably, this molecule is in the form of a plasmid,which may contain other non-viral sequences, such as those for markergenes. This molecule does not contain the AAV ITRs and generally doesnot contain the AAV packaging sequences. To avoid the occurrence ofhomologous recombination, other virus sequences, particularly those ofadenovirus, are avoided in this plasmid. This plasmid is desirablyconstructed so that it may be stably transfected into a cell.

Although the molecule providing rep and cap may be transientlytransfected into the host cell, it is preferred that the host cell bestably transformed with sequences necessary to express functionalrep/cap proteins in the host cell, e.g., as an episome or by integrationinto the chromosome of the host cell. Depending upon the promotercontrolling expression of such stably transfected host cell, the rep/capproteins may be transiently expressed (e.g., through use of an induciblepromoter).

The methods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. For example, the rAAVmay be produced utilizing a triple transfection method using either thecalcium phosphate method (Clontech) or Effectene reagent (Qiagen,Valencia, CA), according to manufacturer=s instructions. See, also,Herzog et al, 1999, Nature Medic., 5(1):56-63, for the method used inthe following examples, employing the plasmid with the transgene,CPA-RPE65, a helper plasmid containing AAV rep and cap, and a plasmidsupplying adenovirus helper functions of E2A, E4Orf6 and VA. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

The rAAV virions are then produced by culturing a host cell containing arAAV virus as described herein which contains a rAAV construct to bepackaged into a rAAV virion, an AAV rep sequence and an AAV cap sequenceunder the control of regulatory sequences directing expression thereof.Suitable viral helper genes, e.g., adenovirus E2A, E4Orf6 and VA, amongother possible helper genes, may be provided to the culture in a varietyof ways known to the art, preferably on a separate plasmid. Thereafter,the recombinant AAV virion which directs expression of the transgene isisolated from the cell or cell culture in the absence of contaminatinghelper virus or wildtype AAV.

One may easily assay whether a particular expression control sequence issuitable for a specific transgene, and choose the expression controlsequence most appropriate for expression of the desired transgene. Forexample, a target cell may be infected in vitro, and the number ofcopies of the transgene in the cell monitored by Southern blotting orquantitative polymerase chain reaction (PCR). The level of RNAexpression may be monitored by Northern blotting or quantitative reversetranscriptase (RT)-PCR; and the level of protein expression may bemonitored by Western blotting, immunohistochemistry, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specificmethods detailed below in the examples.

In one embodiment exemplified below, a suitable recombinant vector foruse in this invention is AAV-RPE65, which utilizes AAV serotype 2 ITRand capsid sequences and is described in detail in Example 1 below. Thisrecombinant AAV contains a CMV immediate early enhancer/chicken β-actin(CβA) promoter-exon 1-intron 1 element followed by a poliovirus internalribosome entry sequence (IRES), followed by the cDNA encoding thewildtype protein RPE65. However, the present invention is not limited tothis exemplary embodiment. Similar rAAV with different transgenes,promoters, IRES, and virus capsids may be useful in this invention, asdescribed in detail above.

D. Pharmaceutical Compositions and Methods of the Invention

The recombinant AAV containing the desired transgene and cell-specificpromoter for use in the target ocular cell as detailed above ispreferably assessed for contamination by conventional methods and thenformulated into a pharmaceutical composition intended for subretinalinjection. Such formulation involves the use of a pharmaceuticallyand/or physiologically acceptable vehicle or carrier, particularly onesuitable for subretinal injection, such as buffered saline or otherbuffers, e.g., HEPES, to maintain pH at appropriate physiologicallevels. A variety of such known carriers are provided in InternationalPatent Publication No. WO 00/15822, incorporated herein by reference. Ifthe virus is to be stored long-term, it may be frozen in the presence ofglycerol.

According to the method of this invention for treating an oculardisorder characterized by the defect or absence of a normal gene in theocular cells of a human or animal subject, the pharmaceuticalcomposition described above is administered to the subject having such ablinding disease by subretinal injection. The use of subretinalinjection as the route of delivery is a critical component of thismethod, as intravitreal administration does not enable the sametherapeutic effects. The vector and carrier cannot diffuse across themultiple cell layers in the retina to reach the RPE, when intravitrealinjection is used. Similarly, intravenous delivery is unacceptablebecause the material does not penetrate the blood-brain (blood-retinal)barrier. Because the virus does not diffuse well, topical administrationis similarly not preferred for this method. See the examples below.

An effective amount of a recombinant adeno-associated virus carrying anucleic acid sequence encoding the desired transgene under the controlof the cell-specific promoter sequence desirably ranges between about1×10⁹ to 2×10¹² rAAV infectious units in a volume of between about 150to about 800 μl. The rAAV infectious units are measured as described inS. K. McLaughlin et al, 1988 J Virol., 62:1963. More desirably, aneffective amount is between about 1×10¹⁰ to 2×10¹¹ rAAV infectious unitsin a volume of between about 250 to about 500 μl. Still other dosages inthese ranges may be selected by the attending physician, taking intoaccount the physical state of the subject, preferably human, beingtreated, the age of the subject, the particular ocular disorder and thedegree to which the disorder, if progressive, has developed.

It may also be desirable to administer multiple “booster” dosages of thepharmaceutical compositions of this invention. For example, dependingupon the duration of the transgene within the ocular target cell, onemay deliver booster dosages at 6 month intervals, or yearly followingthe first administration. The fact that AAV-neutralizing antibodies werenot generated by administration of the rAAV vector, as discussed in theexamples below, should allow additional booster administrations.

Such booster dosages and the need therefor can be monitored by theattending physicians, using, for example, the retinal and visualfunction tests and the visual behavior tests described in the examplesbelow. Other similar tests may be used to determine the status of thetreated subject over time. Selection of the appropriate tests may bemade by the attending physician. Still alternatively, the method of thisinvention may also involve injection of a larger volume ofvirus-containing solution in a single or multiple infection to allowlevels of visual function close to those found in wildtype retinas.

As is demonstrated in the examples below, an exemplary rAAV RPE65 wasemployed in in vitro and in vivo experiments to provide evidence of theutility and efficacy of the methods and compositions of this invention.The in vitro examples demonstrated proper expression of the transgene inan animal model of a human ocular disorder resulting in blindness. Thein vivo examples demonstrated restoration of visual function and visualbehavior by the method of this invention in a large animal model of ahuman retinopathy. The use of the exemplary vector demonstrated in bothin vitro and in vivo experiments that the defect in the RPE65 mutant dogcould be corrected by gene delivery. Vision was restored in this largeanimal model of childhood blindness. This is the first successfulreversal of vision loss in a large animal model of retinal degeneration.This data allow one of skill in the art to readily anticipate that thismethod may be similarly used in treatment of LCA or other types ofretinal disease in other subjects, including humans.

While previous studies have demonstrated that retinal degeneration canbe retarded with gene therapy techniques, the present inventiondemonstrates a definite recovery of function. In addition, while smallanimal studies have demonstrated histologic and electrophysiologiccorrelates of visual function to be partially preserved, this largeanimal study has shown the presence of vision with regard to bothphysiological and behavioral measures.

E. Examples

As summarized, the following examples demonstrate that in three of threeeyes, in vivo transfer of wildtype RPE65 to cells of the outer retinawas sufficient to restore photoreceptor function in the RPE65 mutantdog. Function was not restored after intravitreal injection of vector, aroute which normally results only in transduction of ganglion cells(Dudus, L., et al. Vision Res. 39, 2545-2554 (1999)). The virustransduced RPE cells in the immediately injected quadrant, andtransduced RPE produced both wildtype RPE65 message and protein. Withoutbeing bound by theory, the inventors believe that although the rAAVvirus targets photoreceptors and other retinal neurons as well as RPEcells, and the CβA promoter is active in all these cell types, it islikely that it is the expression of the wildtype transgene in RPE cells(and not photoreceptors) that rescues the mutant phenotype. The RPEalone is responsible for, and possesses the components necessary tosupply chromophore for, rod photoreceptors, although the existence of aretinal retinoid metabolism for cones (not involving RPE65 gene product)remains plausible.

The following examples illustrate several embodiments of this invention.These examples are illustrative only, and do not limit the scope of thepresent invention.

EXAMPLE 1 Virus Preparation

Recombinant AAV vector was based on pTR-UF2, a vector using the 472 bymouse rod opsin promoter to drive expression of green fluorescentprotein (GFP) (Flannery, J., et al. 1997 Proc Natl Acad Sci USA 94,6916-6921). To generate the recombinant vector, AAV-RPE65, the opsinpromoter in pTR-UF2 was replaced with a CMV immediate early enhancer(381 bp)/chicken β-actin (CβA) promoter-exon 1-intron 1 (1352bp) elementfollowed by a poliovirus internal ribosome entry sequence (637 bp). Thelatter supports expression in photoreceptors, RPE and ganglion cells (Liand Hauswirth, unpublished data, 2000). The reporter/transgene GFP wasreplaced with the canine RPE65 cDNA (Aguirre, G. et al, 1998 Mol. Vis.4: 23) via flanking Not I sites and the orientation and reading frameconfirmed by DNA sequence analysis. Plasmid DNA containing thisconstruct was packaged into AAV particles employing iodixanol gradientpurification followed by heparin-sepharose agarose column chromatographyas described in Hauswirth, W. W. et al, 2000 Meth. Enzymol. 316,743-761. Vector titers were determined using an infectious center assay.

Four AAV-RPE65 virus preparations were made and combined to a totalvolume of 1.05 ml at 2.3×10¹¹ infectious particles/ml. Contaminatinghelper adenovirus and wild-type AAV, assayed by serial dilutioncytopathic effect or infectious center assay, respectively were lessthan six orders of magnitude lower than vector AAV.

EXAMPLE 2 In Vitro Testing of an Aav Carrying the Wildtype Canine Rpe65Cdna

A. RPE Cell Cultures

RPE cells from eyes of both a wildtype dog and a homozygous affected(RPE65 mutant) dog were dissociated with 0.25% trypsin (Ray, J. et al,1997 Curr. Eye Res. 16: 131-143) and plated at 1-2×10⁵/9 mm plasticdish. The cells were then cultured. After 48 days, confluent RPEcultures were trypsinized, subcultured and infected at 80% confluencywith 2.3×10⁷ AAV-RPE65 viral particles for 4 hours. Expression of theRPE65 transgene was assessed by immunohistochemistry 10 dayspost-infection.

B. RPE65 Immunocytochemistry and Western Analysis in Canine RPE Cellsand Retina.

In order to evaluate the presence of the RPE65 protein, the culturedcanine RPE cells were evaluated by immunocytochemistry byimmunolabelling with a rabbit anti-RPE65 peptide polyclonal antibody(generously provided by T. M. Redmond) and the nuclei were stained withpropidium iodide. For Western analysis, proteins from cultured RPE wereelectrophoresed on 12.5% SDS-polyacrylamide gel and thenelectrotransferred on nitrocellulose membrane. Immunodetection wasperformed using the anti-RPE65 antibody followed by goat anti-rabbitsecondary antibody and ¹²⁵I-protein A (Verdugo, M., et al. 1998 InvestOphthalmol Vis Sci 39, S719).

In the resulting immunohistochemical sections (not shown) wildtyperetinal cells labeled uniformly and intensely with the anti-RPE65antibody, i.e., they possessed high levels of RPE65. In contrast, RPE65labeling (i.e., RPE protein) was absent in untreated RPE65 mutant cells,60 days in culture, prior to infection with AAV-RPE65, showing onlybackground autofluorescence. Further, lipid inclusions were apparent inthe diseased RPE cells. However, within 10 days of infection of theRPE65 mutant cells (60 days in culture) with AAV-RPE65, the majority ofcells labeled positively with the anti-RPE65 antibody, indicatingpresence of wildtype RPE65 protein. One cell did not appear to have beentransduced. Complementary results were observed followingimmunohistochemistry of sections from untreated wildtype versus mutantRPE65+canine retinas.

Infection of the defective RPE cells by AAV-RPE65 and subsequentexpression of the wildtype RPE65 transgene were further confirmed invitro using PCR amplification and Western analysis, respectively.

PCR studies took advantage of the difference in size of the wildtypeversus mutant canine RPE65 transcripts due to the 4bp deletion in thelatter. PCR amplification utilized RPE65-1 (forward) and RPE65-3(reverse) primers flanking the RPE65 mutant deletion site (Aguirre, G.et al, 1998 Mol. Vis. 4:23). PCR conditions were 94° C. (30 seconds),60° C. (30 seconds), and 72° C. (1 minute) for 34 cycles. PCR productswere separated on a 6% polyacrylamide gel. AAV-RPE65 was used aspositive control. This protocol was also used for PCR screening forshedding virus.

The PCR primers flanking this region amplified the wildtype 109 by RPE65DNA fragment in transduced RPE cells from an affected dog.Non-transduced RPE from the same animal yielded only mutant DNA (105 bp)and normal RPE yielded only the wildtype allele (109 bp). Expression ofwildtype RPE65 in transduced RPE cells from an affected animal was alsoapparent by Western blot analysis of cell lysates. RPE65 expression wasdetected only in the transduced RPE cells; not in uninfected cells.

EXAMPLE 3 In Vivo Studies in the Rpe65 Mutant Dog

A. Ocular Delivery

Effects of intraocular delivery of AAV-RPE65 were studied in four RPE65mutant dogs. For in vivo studies, virus was delivered subretinally orintravitreally under direct surgical visualization using methodsdescribed previously (Bennett, J., et al. 1999 Proc. Natl. Acad. Sci.USA 96, 9920-9925 and Bennett, J. et al, 2000 Meth. Enzymol. 316,777-789). Five eyes from three dogs (BR29, BR33 and BR47) were injectedeither subretinally or intravitreally with AAV-RPE65; the sixth eye wasuntreated (Table 1). The fourth dog (BR46) was maintained as anuntreated control.

Each 150-200 μl subretinal injection of vector (at a concentration of2.3×10¹¹ infectious particles/ml) created a retinal detachment elevatingapproximately 35% of the total retinal area. In 2 eyes (BR33 and BR47)this detachment primarily occupied the nasal-inferior quadrant; in the3rd eye (BR29) the site was temporal-superior. These detachmentsresolved spontaneously within 24 hours. Animals were evaluatedpost-operatively for evidence of ocular or systemic toxicity, virusexposure to extralocular tissue, virus shedding, unfavorable immuneresponse or other untoward effects. As discussed in detail below, nonewas found.

B. Detection of Inflammation

Eyes were evaluated clinically at regular intervals following thesurgery to identify inflammation. Humoral and intraocular antibodiesspecific to AAV capsid proteins were evaluated as described in Bennett,J., et al. 1999 Proc. Natl. Acad. Sci. USA 96, 9920-9925, incorporatedherein by reference. Post-operatively, there was no evidence of ocularor systemic toxicity, or other untoward effect. Hematology and bloodchemistries revealed no evidence of systemic toxicity. Evaluation ofhumoral response prior to and post treatment revealed slightly elevatedanti-AAV capsid titers in pre-treatment serum samples, suggestingprevious exposure to AAV proteins. Antibody titers were increased in twoof the three dogs one month after exposure and in all three dogs 4months after exposure. Non-neutralizing serum antibodies directedagainst RPE65 protein also increased after intraocular exposure toAAV-RPE65.

C. Transgene Expression and Persistence

To correlate transgene expression with changed visual function, onesubretinally injected eye (BR29, right eye) was surgically enucleated 99days post injection. The eyecup was divided into temporal-superior,temporal-inferior, nasal-superior, and nasal-inferior quadrants. Fromeach quadrant, the retina, and the pooled RPE-plus-choroid tissues, wereseparately harvested and dissected under RNase free conditions andrapidly frozen. Total RNA was prepared from retina and RPE/choroid usingthe TRIzol Reagent kit (Life Technologies, Gaithersburg, Md.). DNA wasextracted from the same tissues according to the vendor's protocol. cDNAwas amplified from total RNA using RNA PCR kit (Perkin Elmer, FosterCity, Calif.) and the conditions listed above.

RPE65 expression in neural retina, RPE/choroid, and cultured RPE cellswere detected. Genomic PCR demonstrates persistence of transferred viralDNA in neural retina and RPE-choroid from the injected temporal-superiorquadrant. In other quadrants, the host DNA amplified preferentially andthe viral DNA amplification product is below detectable levels. Fromnoninfected RPE of the affected dog, only mutant product amplifies, but10 days posttransfection in vitro the normal transgene yields theoverwhelming product.

RT-PCR (figures not shown) demonstrated expression of wildtype messagein neural retina from all 4 quadrants, but in RPE-choroid from theinjected quadrant only. Where both products amplify, additional bandsrepresenting heteroduplexes are also seen. The transfected RPE/choroidfrom the injected quadrant expressed higher levels of the transferredcDNA than of the mutant host gene. This was not so in other quadrants.Although transfection of neural retina led to expression of the wildtypeallele in all quadrants, a gradient was present in the relativeintensities of the two alleles among quadrants. In the injectedquadrant, the wildtype allele yielded a much more intense band than thehost mutant allele. From the quadrant below this, the two bands wereapproximately equal in intensity. In the nasal half of the eye, themutant band predominated.

Western analysis demonstrated absence of RPE65 protein in mutant RPEcells prior to transfection, but presence of the protein afterwards.Proteins were labeled with anti-RPE65 antibody.

By PCR analyses of serum and tear fluid, there was no sign of virusshedding at 1 month after injection (data not shown). Reversetranscriptase (RT)-PCR on sera, conjunctiva, eyelids, the gland of thethird eyelid, and the optic nerve from the enucleated eye of BR29 werenegative for the transgene at 103 days post injection, indicating thatvirus escape to extraocular tissues was below detectable levels.

D. Retinal/Visual Function Testing

-   -   1. Electroretinograms (ERGS)

The physiological consequences of the treatments were assessed byelectroretinography (ERG) (Banin, E., et al. 1999 Neuron 23, 549-57).Dogs were dark-adapted (overnight), premedicated with acepromazine (0.55mg/kg, IM) and atropine (0.03 mg/kg, IM) and anesthetized byintermittent ketamine (15 mg/kg, IV, repeated every 15 minutes). Pulserate, oxygen saturation and temperature were monitored throughout. Thecornea was anesthetized with topical proparacaine HCl (1%) and pupilsdilated with cyclopentolate (1%) and phenylephrine (2.5%).

Full field ERGs were recorded using a computer-based system (EPIC-XL,LKC Technologies, Inc., Gaithersburg, Md.) and Burian-Allen contact lenselectrodes (Hansen Ophthalmics, Iowa city, Iowa) (Banin, E., et al. 1999Neuron 23, 549-57). Dark-adapted luminance-response functions wereobtained with blue (Wratten 47A) flash stimuli spanning ˜6 log units(−2.9 to +2.8 log scot-cd.s.m⁻²).

ERG b-wave amplitudes were measured conventionally from baseline ora-wave trough to positive peak; a-wave amplitude was measured frombaseline to negative peak at the maximal stimulus. For isolating conepathway function, dogs were light-adapted and 29 Hz flicker ERGs evokedwith white flash stimuli (0.4 log cd.s.m⁻²) on a background (0.8 logcd.m⁻²); amplitudes were measured between successive negative andpositive peaks and timing from stimulus to the next positive peak.Ocular axial length and pupil diameter were measured for each experimentto permit calculation of retinal illuminance.

The restoration of retinal/visual function in RPE65 mutant dogs bysubretinal AAV-RPE65 was demonstrated by the results of theabove-described ERGs. A comparison of dark-adapted ERGs evoked byincreasing intensities of blue light stimuli in a control dog with ERGsto the same stimuli in RPE65 mutant dog BR33 showed the affected animalhad elevated thresholds, reduced amplitudes and waveform shape changes(i.e., b-waves but no detectable a-waves). Over a 5 log unit range ofincreasing stimulus intensity, the ERG of normal dogs responded withincreasing amplitude of bipolar cell (b-wave) and photoreceptor (a-wave)components. At all intensities these signals were dominated by rodphotoreceptor retinal pathways. Compared to normal dogs, the thresholdstimulus required to elicit an ERG response from 4 month old RPE65mutant dogs was elevated by over 4.5 log units.

Retinal function was dramatically improved in eyes treated withsubretinal AAV-RPE65, compared to pre-treatment recordings. Aftersubretinal AAV-RPE65 therapy, the mutant dog showed a vastly improvedb-wave threshold, a large increase of a- and b-wave amplitudes (althoughnot to normal levels) and an ERG waveform shape that is similar tocontrols. Responses from the right eye of BR33 had b-wave thresholdslower by ˜4 log units than pre-treatment, and appeared similar tonormal.

The details of photoreceptor function were analyzed by the amplitude andtiming of the ERG photoresponses evoked by 2.8 log scot-cd.s.m⁻²flashes. Recordings from three control dogs showed ˜250 μV saturatedamplitudes peaking between 4.5 to 6 ms. Photoreceptor function was nearnoise level in three untreated eyes of RPE65 mutant dogs and two eyestreated with intravitreal AAV-RPE65. Photoresponses (of reducedamplitude but normal timing) were present in all three eyes thatreceived subretinal AAV-RPE65. ERG photoresponses in the threesubretinally injected eyes showed maximal amplitudes of 27, 36 and 58μV, representing ˜16% of normal (mean±SD=246±95 μV; n=7).

Small responses evoked at higher intensities lacked an a-wave. Higherenergy stimuli and recording criteria that elicit, in normal dogs,saturated ERG photoresponses originating from photoreceptors yielded nodetectable signals in affected animals. A flicker ERG response,representing isolated cone pathway function in normals, was absent inaffected animals. Photoresponse amplitudes in subretinally injected eyeswere significantly different (P<0.05) than the amplitudes in untreatedeyes (14±3.4 μV; n=3).

Flicker ERGs in the same eyes as described in the immediately precedingparagraphs demonstrated a lack of detectable cone-mediated responsesfrom RPE65 mutant dogs with untreated or intravitreally treated eyes.All eyes with subretinal AAV-RPE65 treatment recovered cone flickerresponses. Cone flicker ERGs were readily recordable post-treatment;amplitudes ranged from 4 to 6 μV, representing ˜16% of normal (30±8 μV).Intravitreally injected eyes showed no difference from untreated eyesfor all measured ERG parameters.

-   -   2. Pupillometry

Transmission of retinal activity to higher visual pathways wasdemonstrated by pupillometry. Dogs were dark-adapted for more than 3hours and pupil responses were obtained sequentially from each eye usingfull-field green stimuli (−3.2 to +3.0 log scot-cd.m⁻²) of ˜2 secondduration. Pupils were imaged with a video camera under infraredillumination and continuously recorded on a VCR. Dynamic changes inpupil diameters were measured from single frames displayed on the videomonitor in relation to the timing of each stimulus. Pupil responses werecalculated by subtracting the smallest pupil diameter achieved within 2seconds after the stimulus onset from the diameter measured in the dark.

All tested pupils constricted in response to high intensity stimuli.Pupil response as a function of stimulus intensity showed 3.8 log unitelevation of threshold (1 mm response criterion) in untreated eyes (n=3;two eyes of BR46 and one eye of BR29) compared to normal eyes (n=3).Eyes treated with subretinal AAV (n=2; BR33 and BR47) had 0.8 log unitlower thresholds compared to untreated eyes.

A change in pupil diameter was noted in response to 2.5 log cd.m⁻² greenstimulus in one eye of three representative dogs; untreated (BR46),subretinal AAV treated (BR33) and a normal control.

At a suprathreshold intensity, pupillary constriction was greatest innormal dogs and least in untreated RPE65 mutant dogs.Subretinally-treated eye of BR33 responded midway between normal anduntreated. The threshold intensity to reach a criterion pupillaryresponse was improved in subretinally-treated eyes compared withuntreated eyes.

Consistent with ERG and pupillometry results, at 104 dayspost-treatment, flash evoked visual cortical potentials to a series ofincreasing intensities of blue light (Wratten 47) in the dark-adaptedstate and recorded definite waveforms from the subretinally-treated eye.In contrast there were no consistent waveforms at any intensity from theeye treated intravitreally.

-   -   3. Behavioral Testing

Qualitative visual assessment of the 3 treated animals was performed at4 months post injection using an obstacle course and observers masked tothe experimental design. Visual behavior was also documented by videorecording. Results of behavioral testing were consistent with theelectrophysiological results.

For example, dog BR33 was consistently (5/5 observers) scored as“normally sighted” under photopic (room lighting) conditions. Under dimred light this dog consistently avoided objects either directly in frontof her, or to her right (the side injected subretinally), butconsistently failed to avoid objects on the left (injectedintravitreally). In contrast, the untreated control affected dog, BR46walked into objects ahead of her and at either side.

Table 1 provides the data collected from the procedures performed on theeyes of four RPE65 mutant dogs. In the Table, age is recorded as dayspostnatal. The abbreviation Rt is used for right eye, while left isindicated for left eye. The routes of injection are identified as SR forsubretinal injection, IV for intravitreal injection, and NI for notinjected. The doses are reported as No.×10¹⁰ infectious particles ofrecombinant AAV-RPE65 virus injected. Baseline ERGs were recorded 2weeks prior to injection. Rescue Effect was assessed by ERGs recorded 95days after injection. Positive effect is indicated by POS. NEG indicatesno effect apparent.

TABLE 1 ERG Age at Route of Volume Age at Rescue Animal Day 0 EyeInjection Dose (μL) ERG Effect BR29 132 Rt SR 3.7 150 227 POS Left NI —— 227 NEG BR33 124 Rt SR 4.6 200 219 POS Left IV 4.6 200 219 NEG BR47108 Rt SR 4.6 200 203 POS Left IV 4.6 200 203 NEG BR46 108 Rt NI — — 203NEG Left NI — — 203 NEG

All references and documents disclosed above are incorporated byreference herein. Numerous modifications and variations of the presentinvention are included in the above-identified specification and areexpected to be obvious to one of skill in the art. Such modificationsand alterations to the compositions and processes of the presentinvention are believed to be encompassed in the scope of the claimsappended hereto.

1. A method for treating an ocular disorder characterized by a defect orabsence of a normal gene in ocular cells of a human subject, the methodcomprising the step of administering to said subject by subretinalinjection a recombinant adeno-associated virus (rAAV) comprising anucleic acid sequence encoding a normal gene under the control of apromoter sequence which expresses the product of the gene in ocularcells, wherein expression of the normal gene provides to the cells theproduct necessary to restore or improve visual function in said subject,wherein said normal gene is the LCA5 or RP12 gene.
 2. A composition fortreatment of an ocular disorder characterized by a defect or absence ofa normal gene in ocular cells of a subject, said composition comprisingt of a recombinant adeno-associated virus (rAAV) carrying a nucleic acidsequence encoding said normal gene under the control of a promotersequence which expresses the product of said normal gene in said ocularcells, formulated with a carrier and additional components suitable forsubretinal injection,. wherein said normal gene is the LCA5 or RP12 gene3. The method according to claim 1, wherein the ocular disorder is Lebercongenital amaurosis (LCA) and said normal gene is LCA5.
 4. The methodaccording to claim 3, wherein said LCA is caused by a mutation in thenormal LCA5 gene.
 5. The method according to claim 1, wherein the oculardisorder is retinitis pigmentosa (RP) and said normal gene is the RP12gene.
 6. The method according to claim 5, wherein the RP is caused by amutation in the RP12 gene.
 7. The method according to claim 1, whereinsaid ocular cells are selected from the group consisting of RPE cellsand photoreceptor cells.
 8. The method according to claim 1, whereinsaid promoter is cell-specific or comprises a chicken beta actinpromoter/CMV enhancer.
 6. The method according to claim 1, wherein saidnormal gene is obtained from the same subject species as the subjectbeing treated.
 7. The method according to claim 1, wherein said rAAVvector is administered in a physiologically acceptable vehicle.
 8. Themethod according to claim 7, wherein said vehicle comprises one or moreof a buffer and a surfactant.
 9. The method according to claim 1,wherein said normal gene is obtained from the same subject species asthe subject being treated.
 10. The composition according to claim 2,wherein the ocular disorder is Leber congenital amaurosis (LCA) and saidnormal gene is LCA5.
 11. The composition according to claim 10, whereinsaid LCA is caused by a mutation in the normal LCA5 gene.
 12. Thecomposition according to claim 2, wherein the ocular disorder isretinitis pigmentosa (RP) and said normal gene is the RP12 gene.
 13. Thecomposition according to claim 12, wherein the RP is caused by amutation in the RP12 gene.
 14. The composition according to claim 2,wherein said ocular cells are selected from the group consisting of RPEcells and photoreceptor cells.
 15. The composition according to claim 2,wherein said promoter is cell-specific or comprises a chicken beta actinpromoter/CMV enhancer.
 16. The composition according to claim 2, whereinsaid normal gene is obtained from the same subject species as thesubject being treated.
 17. The composition according to claim 2, whereinsaid rAAV vector is administered in a physiologically acceptablevehicle.
 18. The composition according to claim 17, wherein said vehiclecomprises one or more of a buffer and a surfactant.
 19. The compositionaccording to claim 2, wherein said normal gene is obtained from the samesubject species as the subject being treated.